In some fields of industry such as drug manufacturing, a familiar technology is a method for production of a recombinant protein of interest using mammalian cells transformed with an expression vector that contains an incorporated gene encoding the protein. Using this technology, various products are produced and marketed, e.g., lysosomal enzymes such as α-galactosidase A, iduronate-2-sulfatase, glucocerebrosidase, galsulfase, α-L-iduronidase, α-glucosidase, and the like; tissue plasminogen activator (t-PA); blood coagulation factors such as blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, and the like; erythropoietin; interferon; thrombomodulin; follicle-stimulating hormone; granulocyte colony-stimulating factor (G-CSF); various antibody medicaments, and the like.
In performing this technology, it is a general practice to employ an expression vector in which a gene encoding a protein of interest is incorporated downstream of a gene regulatory site that induces a potent expression of a gene, such as a cytomegalovirus (CMV)-derived promoter, SV40 early promoter (SV40 enhancer/promoter), or elongation factor 1α (EF-1) promoter. Mammalian cells, after introduction therein of such an expression vector, come to express the protein of interest that is incorporated in the expression vector. The levels of its expression, however, vary and are not even among those cells. Therefore, for efficient production of the recombinant protein, a step is required to select, from the mammalian cells containing the expression vector introduced therein, those cells which express the protein of interest at high levels. For performing this selection step, a gene that acts as a selection marker is incorporated in an expression vector.
The most popular of such selection markers are enzymes (drug resistance markers) that decompose drugs such as puromycin, neomycin, and the like. Mammalian cells will be killed in the presence of these drugs beyond certain concentrations. Mammalian cells into which an expression vector has been introduced, however, become viable in the presence of those drugs because such cells can decompose the drugs with the drug selection markers incorporated in the expression vector and thus detoxify them or weaken their toxicity. Therefore, when those cells having such an incorporated expression marker are cultured in a medium containing one of the above mentioned drugs beyond a certain concentration, only such cells grow that express the corresponding selection marker at high levels, and as a result, they are selected. Such cells that express a drug selection marker at high levels also tend to express, at high levels, a gene encoding a protein of interest incorporated together in the expression vector, and as a result, mammalian cell thus will be obtained that express the protein of interest at high levels.
There is also known a method to obtain mammalian cells that express a protein of interest at high levels utilizing dihydrofolate reductase (DHFR) as a selection marker (Non-patent Document 1). Dihydrofolate reductase is an enzyme which reduces dihydrofolate to tetrahydrofolate. Mammalian cells will die if they are cultured in a thymidine-hypoxanthine-free medium in the presence of methotrexate (MTX), an inhibitor of DHFR, beyond a certain concentration. However, if an expression vector containing an incorporated DHFR gene as a selection marker is introduced into mammalian cells, they become capable of growing even at higher concentrations of MTX because of elevated expression levels of DHFR in them. In this circumstance, if culture is continued gradually elevating the MTX concentration, such cells are obtained that can grow in the presence of even higher concentrations of MTX. This phenomenon is thought to occur because of increase in number of the copies of the expression vector incorporated into the genome of the mammalian cells by multiplication. That is, an increase in number of copies of the expression vector leads to a corresponding increase in number of the DHFR genes in the genome of each cell, resulting in relatively enhanced levels of expression of DHFR. In this process, the number of copies of the gene encoding a protein of interest and simultaneously incorporated in the expression vector also increases, and thus gives mammalian cells that express the protein of interest at high levels.
Expression vectors are also known in which a glutamine synthetase (GS) is used as a selection marker (cf. Patent Documents 1 and 2). Glutamine synthetase is an enzyme which synthesizes glutamine from glutamic acid and ammonia. If mammalian cells are cultured in a medium which lacks glutamine in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, beyond a certain concentration, the cells will be annihilated. However, if an expression vector into which a glutamine synthetase has been incorporated as a selection marker is introduced into mammalian cells, the cells, now with increased levels of expression of the glutamine synthetase, become capable of growing even in the presence of higher concentrations of MSX. In doing this, if culture is continued with a gradually increasing concentration of MSX, such cells are obtained that can grow in the presence of still higher concentrations of MSX. This phenomenon occurs in the same mechanism as where DHFR is used as a selection marker. Therefore, by incorporating in an expression vector a gene encoding a protein of interest together with a GS gene, such mammalian cells will be obtained that express the protein of interest at high levels. For example, Patent Document 1 discloses that by employment of a GS gene and methionine sulfoximine (MSX) enables greater increase of the copy number of the vector DNA than where DHFR gene and methotrexate (MTX) are employed. Further, Patent Document 2 discloses that by employment of a GS gene and MSX, the copy number of a different, heterozygous gene can also be increased, along with increased number of copies of the GS gene, which thereby enables increased production levels of a polypeptide of interest.
Thus, expression vectors containing a selection marker are suitable for efficient production of recombinant proteins, and thus are commonly used. A gene encoding a protein of interest and a gene encoding a selection marker are generally incorporated in an expression vector downstream of respective different gene regulatory sites (cf. Patent Document 3). However, a method is also known in which genes encoding a protein of interest and a selection marker are incorporated in series downstream of a single gene regulatory site to let them express themselves (cf. Patent Documents 4-7). In performing this, an internal ribosome entry site (IRES) and the like are inserted between the genes encoding a protein of interest and a selection marker, which enables expression of two genes under a single gene regulatory site. Various internal ribosome entry sites are known: for example, those derived from picornavirus, poliovirus, encephalomyocarditis virus, and chicken infectious Fabricius bursal disease virus (cf. Patent Documents 8-10).
Among expression vectors utilizing an internal ribosome entry site, there are known an expression vector in which herpes simplex virus thymidine kinase is incorporated as a selection marker downstream of an internal ribosome entry site (cf. Patent Document 11), and an expression vector in which three or more genes are combined using two or more internal ribosome entry sites (cf. Patent Document 12).
As mentioned above, owing to development of various expression vectors, methods for production of recombinant proteins using mammalian cells have been in practical use for production of medicaments, such as erythropoietin and the like. However, development of expression vectors which are more efficient than conventional ones are consistently sought in order to lower the cost for their production.